Novel PEGylated cholephytosomes for targeting fisetin to breast cancer: in vitro appraisal and in vivo antitumoral studies

Abstract

Fisetin (FIS) is a multifunctional bioactive flavanol that has been recently exploited as anticancer drug against various cancers including breast cancer. However, its poor aqueous solubility has constrained its clinical application. In the current work, fisetin is complexed for the first time with soy phosphatidylcholine in the presence of cholesterol to form a novel biocompatible phytosomal system entitled "cholephytosomes." To improve fisetin antitumor activity against breast cancer, stearylamine bearing cationic cholephytosomes (mPHY) were prepared and furtherly modified with hyaluronic acid (HPHY) to allow their orientation to cancer cells through their surface exposed phosphatidylserine and CD-44 receptors, respectively. In vitro characterization studies revealed promising physicochemical properties of both modified vesicles (mPHY and HPHY) including excellent FIS complexation efficiency (˷100%), improved octanol/water solubility along with a sustained drug release over 24 h. In vitro cell line studies against MDA-MB-231 cell line showed about 10- and 3.5-fold inhibition in IC50 of modified vesicles compared with free drug and conventional drug-phospholipid complex, respectively. Preclinical studies revealed that both modified cholephytosomes (mPHY and HPHY) had comparable cytotoxicity that is significantly surpassing free drug cytotoxicity. TGF-β1and its non-canonical related signaling pathway; ERK1/2, NF-κB, and MMP-9 were involved in halting tumorigenesis. Thus, tailoring novel phytosomal nanosystems for FIS could open opportunity for its clinical utility against cancer.

Keywords: Breast cancer; Cholephytosomes; Fisetin; Hyaluronic; Stearylamine.

Fig. 1

Effect of the gradual titration of anionic biopolymer (hyaluronic acid; HA) on zeta potential of optimized cationic FIS-PEGylated cholephytosomes (mPHY)

Fig. 2

TEM photomicrographs of mPHY (a) and HPHY (b) at magnification = 50,000 × . Scale bar = 100 nm

Fig. 3

In vitro release profiles of free FIS solution (SOL), FIS suspension (SUS), cationic FIS-PHY (mPHY), and HA-coated phytosomes (HPHY) using dialysis method in phosphate-buffer saline (PBS) pH 7.4 containing 20% alcohol at 100 rpm and 37 °C. Results are expressed as mean ± SD (n = 3); some error bars are too small to be presented

Fig. 4

Viability assay of free fisetin (D), physical mixture of FIS and SPC (FM), conventional FIS-phytosomes (PLX), cationic fisetin cholephytosomes (mPHY), blank cationic nanovesicles (B-mPHY), and HA-coated FIS-cholephytosomes (HPHY) after incubation for 24 h with MDA-MB-231 breast cancer cells

Fig. 5

IC50 in µg/ml for free FIS (D) and different FIS-phospholipid complexes against MDA-MB-231 breast cancer cell line after 24 h. Conventional FIS-phytosomes (PLX), cationic fisetin cholephytosomes (mPHY), and HA-coated FIS-cholephytosomes (HPHY)

Fig. 6

Apoptotic assay for FIS-phytosomes against fisetin solution at 20 µg/ml equivalent dose of FIS using annexin-v-FITC/propidium iodide assay by flow cytometry after incubation for 24 h with MDA-MB-231 cells. Abbreviations; D fisetin solution in DMSO, PLX conventional FIS-phytosomes, mPHY cationic FIS-cholephytosomes, HPHY hyaluronic-coated FIS-cholephytosomes

Fig. 7

Assessment of nuclear morphology by DAPI staining of MDA-MB-231 nuclei using confocal microscope. Photographs of control cells showed normal and uniform nuclear structure with a large and round nucleus and uniform chromatin density. Treated cells with 20 µg/ml of fisetin (F) or equivalent dose of conventional phytosomes (PLX) and modified cholephytosomes (mPHY and HPHY) showed a significant change in nuclear morphology. White arrows manifest fragmented nuclei with chromatin condensation. Incubation time was 24 h, and the scale bar is 25 µm

Fig. 8

Effect of FIS (D) and modified cholephytosomes (mPHY and HPHY) on percent increase of tumor size. PC untreated mice, D FIS solution, mPHY cationic SA-bearing cholephytosomes, and HPHY hyaluronic decorated cholephytosomes. Two-way ANOVA was used to test effect of treatment and followed by Bonferroni test

Fig. 9

Effect of FIS (D) and modified cholephytosomes (mPHY and HPHY) on immunohistochemical expression of TGB-1 in breast carcinoma: representative photomicrograph (TGB-1, X400) (a) and quantitative determination (b) of (I) percent immunostaining and (II) intensity of stain. PC: untreated mice, mPHY: cationic SA-bearing cholephytosomes, HPHY: hyaluronic decorated cholephytosomes. Statistical analysis was done using one-way ANOVA followed by Student–Newman–Keuls multiple comparison test; *P < 0.05 vs PC, $P < 0.05 vs D, λ P < 0.05 vs mPHY

Fig. 10

FIS (D) and modified cholephytosomes (mPHY and HPHY) on: p-ERK1/2 (a), NF-κb (b), MMP9 (c). PC untreated mice, mPHY cationic SA-bearing cholephytosomes, and HPHY hyaluronic decorated cholephytosomes. Statistical analysis was done using one-way ANOVA followed by Student–Newman–Keuls multiple comparison test; *P < 0.05 vs PC, $P < 0.05 vs D, λ P < 0.05 vs mPHY

Fig. 11

Effect of FIS (D) and modified cholephytosomes (mPHY and HPHY) on the immunohistochemical expression of E-cadherin in breast carcinoma: representative photomicrograph (X400) (a) and quantitative determination (b) of (I) percent immunostaining and (II) intensity of stain. PC untreated mice, mPHY cationic SA-bearing cholephytosomes, HPHY hyaluronic decorated cholephytosomes. Statistical analysis was done using one-way ANOVA followed by Student–Newman–Keuls multiple comparison test; *P < 0.05 vs PC, $P < 0.05 vs D